Background/Purpose: RA is the most common autoimmune inflammatory joint disease, affecting up to 1% of the world population. Detection of antibodies, specifically against IgM rheumatoid factor (IgM-RF) and anti-cyclic citrullinated peptide (anti-CCP) is widely used for the diagnosis of RA; both of these antibodies are included in the 2010 ACR/EULAR Rheumatoid Arthritis Classification Criteria. However, novel antibodies have been identified that can help further diagnose seronegative (IgM-RF and anti-CCP negative) RA patients. In addition to antibodies targeting cyclic citrullinated peptide (CCP), antibodies to citrullinated alpha-enolase peptide 1 (CEP-1) and citrullinated vimentin (Sa) have been found in RA synovium and serum. 14-3-3η is a protein also found in high levels in synovial fluid and serum of RA patients. The purpose of this study is to examine the sensitivity and specificity of 14-3-3η, anti-CEP-1 and anti-Sa antibodies in a cohort of seronegative RA and suspected RA patients.

Methods: The 3 assays were analyzed by enzyme-linked immunosorbent assay (ELISA) in serum from a community rheumatology practice in Los Angeles, CA (USA) using 176 samples of which, 130 were established RA patients and 46 suspected RA patients.  Specificity was tested using 20 SLE, 16 psoriatic arthritis, 12 Sjogren’s Syndrome and 20 Non-Rheumatic Disease Patient (NRDP) samples. Levels of 14-3-3η (Augurex Life Sciences Corp.), anti-CEP-1 and anti-Sa antibodies (Euroimmun) where measured using a laboratory developed test (LDT) at Rheumatology Diagnostics Laboratory, Inc. (RDL). Levels of anti-CCP IgG/IgA and IgM-RF were obtained on all serum samples.           

Results: Out of 130 established RA patients, 50 (38%) were seronegative for both IgM-RF and anti-CCP antibodies. Of these 50 seronegative patients, 14-3-3η was positive in 5 patients (10%), while anti-CEP-1 and/or anti-Sa antibodies were positive in 5 additional patients (10%). Out of the 46 suspected RA patients, 38 (83%) were seronegative for both IgM-RF and anti-CCP antibodies. Of these 38 seronegative suspected RA patients, 14-3-3η was positive in 4 patients (11%), while no anti-CEP-1 or anti-Sa antibodies were positive.  

Conclusion:    In an established RA cohort negative for both IgM-RF and anti-CCP antibodies, further testing for 14-3-3η, anti-CEP-1 and anti-Sa antibodies identified an additional 20% of RA patients. In suspected RA patients (seronegative), 14-3-3η was positive in an additional 11%. The overall specificity of the 3 markers was 85%. In the autoimmune disease controls, most of the patients that tested positive for these antibodies had inflammatory arthritis/active synovitis, and therefore may have had an overlap syndrome.